In May 2003, the ion channel community was galvanized by the publication of the high resolution X-ray structure of an archaebacterial voltage-dependent K+ channel, KvAP, by MacKinnon and co-workers. KvAP contains sequence hallmarks and functional properties that establish it as a close relative of eukaryotic voltage-dependent K+ channels. The structure of the pore domain in KvAP is similar to KcsA, MthK, and KirBac. In contrast, the structure of the voltage sensor domain in KvAP contains several surprising features that were unanticipated from previous work. Largely influenced by this structure, MacKinnon and co-workers have proposed a new model for voltage-dependent activation in which the voltage sensor domain acts as a hydrophobic paddle that moves through the lipid bilayer during activation. The KvAP structure and the hydrophobic paddle mechanism have generated significant controversy and refocused attention on three essential questions: What is the structure of the voltage sensor? How does it move during activation? How do voltage sensor movements open and close pore gates? Answering these questions is the long-term goal of this research project. The Specific Aims of the proposal are: 1) to investigate the topology of KvAP and proximity between the voltage sensor and pore domains in a native membrane environment; 2) to investigate the mechanism of voltage-dependent activation in KvAP, 3) to investigate voltage sensor/pore domain interactions in Shaker channels, and 4) to investigate voltage sensor conformational changes in ether-[unreadable]-gogo (eag) using combined electrophysiological and optical measurements. This proposal describes basic research aimed at understanding the structure and function of voltage-dependent ion channels. The research is likely to have significant health relevance because ion channels have essential roles in the brain, heart, and skeletal muscle.